The present invention pertains to solid enzyme preparations, free of surface active agents, containing proteases, obtained by tanning precipitation, as the active enzyme, and to methods for the soaking, bating, and unhairing of hides in the preparation of leather.
The technique of tannin precipitation has been known for a long time as an occasionally used method variant for the isolation of enzymes from solutions, e.g. from plant juices or aqueous culture media. There is a relatively broad state of the art relevant thereto (cf., e.g., GB-C 1,156,900, DE-A 1,642,619). As a rule, the goal is purification of the enzyme. The addition of gelatin, or working in an acid pH region (pH 3-5) serve as measures assisting good precipitation. However, because tannin disrupts further enzyme processingxe2x80x94after all, it forms an insoluble complex with the enzymes, whereupon they precipitatexe2x80x94it must again be removed, which is usually done by treating the precipitate with organic solvents, e.g. with acetone or ethanol, or by the addition of surface active agents, or by an increase in the pH value of the tannin precipitation. These are all measures which generally can be performed only on a laboratory scale without considerable complications.
Technical use of the tannin complex itself has been described in only a few cases. Thus, a tannin complex together with skim milk is said to be worked up by drying into a stable xcex1-amylase preparation (CS 141,233). For medical use, an insoluble pancreas preparation is provided which was prepared by precipitation of pancreas with tannic acid. The preparation is insoluble in acid stomach juice, but develops its effect in the alkaline intestinal regions. (DE 128,419).
Further, DE-A 2,143,945 describes xe2x80x9cwater-insoluble, dried enzyme adducts which are compatible with the skinxe2x80x9d, inter alia in the form of tannin complexes. For practical use, the incorporation directly into a washing powder of an adduct of a bacterial protease obtained by tannin precipitation is proposed, for example. Since the tannin complex is said to be fully effective as a washing agent during use, i.e. in the washing float, it must xe2x80x9cbreak upxe2x80x9d.
As is evident from the foregoing picture of the state of the art, the method of tannin precipitation of enzymes from an aqueous milieu is, to be sure, a useful precipitation technique, but the relative stability of precipitated enzyme-tannin complexes under the conditions prevailing in potential uses, seemsxe2x80x94apart from the exceptions namedxe2x80x94to stand in the way of their industrial utilization.
Heretofore, ammonium sulfate or sodium sulfate have preferably been used for precipitation from the juice of pressed pancreas or from bacterial cultures (cf. Ullmanns Encyclopxc3xa4die der technischen Chemie, 4th edition, volume 10, pages 475-561, page 495 ff., Verlag Chemie, 1975; DE-A 2,234,412).
However, this procedure is by no means idealxe2x80x94particularly from an ecological viewpointxe2x80x94since as a rule it incurs a quite considerable loading of the waste water. This is evident from the rule of thumb which says that 50 kg of ammonium sulfate are necessary for precipitation for each 100 liters of juice containing enzyme. Such a salt load is strictly contrary to the present tendency of waste water technology to restrict sulfate loading as much as possible. Rather, an efficient method for precipitating enzymes was to be strived for, in which the waste water loading would be considerably lessxe2x80x94as well as, also, more cost effective, particularly in view of the quite particular cost pressure under which the leather industry operates. It has now been found that the proteinase preparation according to the invention comes very close to the aforementioned technical conceptions. The invention pertains to a solid, i.e. powdered or granulated, enzyme preparation essentially free of surface active agents and containing a proteasexe2x80x94obtained as a tannin complex by precipitation of the active enzyme from an aqueous medium by the addition of tanninxe2x80x94with the proviso that the enzyme preparation containing at least 50 percent by weight, preferably more than 80 percent by weight, and up to 99.9 percent by weight, of one or more salts conventionally used as extenders or diluent in commercial enzyme preparations.
By xe2x80x9ctanninsxe2x80x9d are meant according to the present invention the polyphenols, as a rule of natural origin, encompassed under this name, particularly the tannic acids. (More exact data are found in Ullmann, Encyclopxc3xa4die der Tech. Chemie, 3rd edition, volume 11, pages 593-594; Kirk-Othmer, Encyclopedia of Chemical Technology, 2nd edition, volume 12, pages 319-325, J. Wiley 1967; Fortschritte der Chemie organischer Naturstoffe, Zechmeister, editor, volume 41, 1-46, Springer Verlag).
By xe2x80x9cproteasesxe2x80x9d are to be understood the enzymes included under E.C.3.4. (Cf. Kirk-Othmer, Encyclopedia of Chemical Technology, 3rd edition, volume 9, pages 173-223, J. Wiley 1980; E. Pfleiderer and R. Reiner in H. J. Rehm and G. Reed, Biotechnology, volume 6b, pages 729-742, VCH 1988; K. Aunstrup in Industrial Aspects of Biochemistry, B. Spencer, editor, volume 30(I), pages 23-46, North Holland 1974).
Various sub-criteria are applied, among them differentiation according to origin:
a) animal origin, such as
xcex1) rennin (E.C.3.4.23.4)
xcex2) pancreas proteases
pancreatin, particularly trypsin; chymotrypsin (pH region of efficacy ca. 7-10); pepsin (E.C.3.4.23.1) (pH region of efficacy ca. 1.5-4.0; cathepsin (E.C.3.4.23.5) (region of efficacy ca. 4.0-5.0)
b) plant origin
xcex1) papain (E.C.3.4.22.1) pH region of efficacy ca. 5.0-8.0
xcex2) ficin (E.C.3.4.22.3) pH region of efficacy ca. 4.0-9.0
xcex3) bromelin (E.C.3.4.22.4 and 3.4.22.5) pH region of efficacy ca. 5.0-7.0
c) microbial origin (cf. L. Keay in Process Biochemistry 1971, pages 17-21)
xcex1) from Bacillus species
such as B.subtilis, B.licheniformis, B.alkalophilus, B.cereus, B.natto, B.vulgatus, B.mycoides 
xcex2) from Streptococcus species
xcex3) from Streptomyces species
such as Streptomyces fradiae, S.qriseus, S.rectus 
xcex4) from Aspergillus species
such as Aspergillus flavus-oryzae, A.niger, A.saitoi, A.usamii 
xcex5) from Mucor and Rhizopus species
such as Mucor pusillus, M.miehei 
xcex6) Endothia species
such as Endothia parasitica 
xcex7) from Trametes species
such as Trametes sanquinea. 
Besides this differentiation according to origin, differentiation according to the kind of attack (exo- versus endo-enzymes) and on the basis of the xe2x80x9cactive sitexe2x80x9d of the proteases (serine proteases, which are inhibited by diisopropylfluorophosphate, sulfhydryl enzymes) is also used.
Further, the pH dependence of the enzyme activity is of considerable practical significance. Here, differentiation is, above all, according to practical criteria.
i) alkaline proteases, having an activity optimum about in the region from pH 7.5 to 13,
particularly alkaline bacterial proteases (E.C.3.4.21.) (which mostly belong to the serine type) and alkaline fungal proteases
ii) neutral proteases, having an activity optimum in the range from pH 6.0-9.0,
particularly neutral bacterial proteases (E.C.3.4.24) (which belong to the metalloenzymes) and fungal proteases, for example Bacillus proteases, Pseudomonas proteases, Streptomyces proteases, Aspergillus proteases
iii) acid proteases, having an activity maximum in the range from pH 2.0-5.0 (E.C.3.4.23)
particularly acid fungal proteases, e.g. from Rhizopus species, Aspergillus species, Penicillium species, Mucor species, and Impex lacteus and Endothia parasitica. 
Subtilisins, alkaline bacterial proteases of the serine type which are stable in the pH region 9-10 and are to some degree insensitive to perborate, are especially mentioned as alkaline proteases.
The use of proteolytic enzymes is an established part of leather manufacture, particularly in the beamhouse, since the introduction of enzymatic bating (tryptic digestive enzymes of the pancreatic gland) in the xe2x80x9cOROPONxe2x80x9d bate by Dr. Otto Rxc3x6hm (DE-PS 200,519) about 80 years ago. In addition to use in the bate (DP-PS 927,464; DE-PS 976,107; DE-PS 941,811; DE-PS 974,813; DE-PS 975,095; DE-PS 976,928; DE-PS 1,120,066;DE-PS 1,134,474; DE-PS 1,219,620; DE-PS 1,282,837; U.S. Pat. Nos. 3,939,040; 4,273,876) enzyme preparations are also used in soaking (DE-PS 288,095; DE-PS 976,662; DE-PS 1,022,748; DE-PS 1,034,317; DE-PS 1,282,828; DE-PS 2,059,453; U.S. Pat. Nos. 4,278,432; 4,344,762), as well as in hair loosening and hide opening (U.S. Pat. No. 4,294,087).
The proteolytic efficacy of enzymes is commonly determined according to the Anson hemoglobin method [M. L. Ansen, J. Gen. Physiol. 22, 79 (1939], or as xe2x80x9cLVExe2x80x9d (Lxc3x6hlein-Volhard Units) according to the Lxc3x6hlein-Volhard method (Die Lxc3x6hlein-Volhard""sche Methode zur Bestimmung der proteolytic Activitxc3xa4txe2x80x9c, Gerberiechem. Taschenbuch, Dresden-Leipzig, 1955). One Lxc3x6hlein-Volhard unit is defined as that amount of enzyme which digests 1.725 mg of casein under the specific conditions of the method (cf. R. J. Beynon, J. S. Bond, Proteolytic Enzymes, IRL Press).
Further in the following, units derived from the Anson method are used for the determination of the activity of enzymes effective in the acid region. These units are designated xe2x80x9cproteinase units (Hemoglobin)xe2x80x9d, UHb. One UHb corresponds to that amount of enzyme which catalyzes the liberation from hemoglobin of fragments, soluble in trichloroacetic acid, equivalent to 1 xcexcmol of tyrosine per minute at 37xc2x0 C. (measured at 280 nm). (1 mUHb=10xe2x88x923 UHb).
As already mentioned, the preparation of the proteinase preparation according to the invention advantageously proceeds directly from aqueous culture media or juices containing enzymes.
The winning of the preparation from the pancreas is of special interest.
A. Winning from the pancreatic complex
The techniques of the invention can partially utilize isolation methods of the prior art (cf. Ullman, op.cit., 4th edition, volume 10, pages 536-537.)
Isolation advantageously proceeds from pancreatic glands directly after slaughter, predominantly of swine or cattle.
For example about 100 pancreatic glands can be worked up in one batch by removing fat and connective tissue from the glands as completely as possible immediately after slaughter and then homogenizing the gland tissue, e.g. using a mincing machine. Immediately thereafter, extraction follows, suitably with about double the volume (about 60 liters) of 0.25 N sulfuric acid at 5xc2x0 C. for 18-24 hours. After the addition of filter flakes, the extract is filtered, preferably using a packing press.
The largely fat-free liquid of the pressings obtained in this way is the starting material for isolation of the protein preparation.
B. Winning from other aqueous crude extracts containing proteinase
Instead of the liquid from pancreas pressings, other protein-containing culture liquids, e.g. of fungal or bacterial cultures, can be used (cf. the prior teachings concerning the source of enzyme as well as Ullmann, op. cit., 4th edition, volume 10, pages 518-522; Biotechnology, H. J. Rehm and G. Reed, editors, volume 7a, pages 156-168, Verlag Chemie 1987).
The culture liquids containxe2x80x94as an approximate reference pointxe2x80x94between 0.01 and 3 percent by weight of protein.
The proteases of the invention can be recovered as solid enzyme products in the form of a powder or granulate containing an effective enzyme and free of surface active agents as follows:
First, the correct dosage of the precipitating agent has proved to be important. If a deficient amount of tannin is used, the precipitation is incomplete; with an excess of tannin, the tannin complex is broken up only with difficulty.
As a convenient technical rule, it has turned out that just so much precipitant should be used that 0.5-3 percent of the enzyme activity originally present before the addition of tannin is still found in the supernatant. Thus, for example, for precipitation from pancreas liquid pressings (containing about 2 percent by weight of active protein), 4 percent by weight of mimosa tannin is used. In general, the protein content in the tannin complexes is from 10 to 80 percent by weight.
More in detail, one advantageously proceeds such that an aqueous liquid having a low pH value is prepared (to be sure, attention must be paid to the stability of the proteases involved). As a reference point, a pH value of about 3-6 is mentioned: Culture or pancreas liquids mostly have a pH value of about 6. Precipitation above pH=7 is less advantageous. In every case, it is advantageous to use low temperatures (20xc2x0 C. or below). Also, the addition of water soluble surface active agents having an HLB value less than 6 has proved favorable. Such surfactants are chosen, for example, from the class of long-chain esters, such as the sorbitan fatty acid esters for instance. Sorbitan monooctadecanoate, for example, can be mentioned inter alia. Such surfactants are commercially available, cf. products of Atlas Chemie, Essen, of the xe2x80x9cSPANxe2x80x9d type, for example.
By definition, a salt content of at least 50 percent by weight is present in the enzyme preparation, consisting of one or more salts conventionally used as diluents. These salts may be, preferably, ammonium sulfate or sodium sulfate, for example.
Advantageous Effects
Advantageous effects are already noted from the point of view of preparation and availability, but not last are such effects in the use of the enzyme preparation of the invention. The preparation of the tannin complexes involves a very good utilization of resources, because it involves both an effective and at the same time selective precipitation method. The considerably lower environmental pollution by the xe2x80x9cmother liquorxe2x80x9d, when compared with the salt precipitations most often performed, is emphasized. The enzyme preparations can be classed as xe2x80x9cadvancedxe2x80x9d from the point of view of work-place hygiene because they are not dust forming and have scarcely any allergenic effect on the skin.
From the point of view of uses, it can be considered an advantage that the enzyme preparations according to the invention can be used within the traditional technical procedures, i.e. there is no basic change necessary in the course of treatment in the beamhouse. The use of the enzyme preparation according to the invention has proved valuable particularly in the course of the traditional enzymatically-assisted soaking and in the bate. In such treatments, the presence of a high pH value during use is particularly advantageous (pH greater than 8) because such favors the xe2x80x9cbreaking upxe2x80x9d of the tannin complexes.
When used in soaking, the preparations of the invention bring about a faster rehydration of salt-conserved hides. When used in bating, the preparations of the invention effect a better removal from the hide surface of pigment residues and hair roots.
Use: Soaking
The soaking of hide material, by which the hardening of the hide which occurs because of salt preservation is reversed, is usually carried out at a pH greater than 7 to 10. The joint use of enzymes, particularly of proteolytic enzymes, accelerates the soaking effect by xe2x80x9cdigestionxe2x80x9d of the water soluble and other protein bodies which do not belong to the collagenous fibrous structure of the hide. In general, in soaking, enzymes having an effective range (or pH optimum of the proteolytic effect) at a pH 7.0-10.0 are used. By the removal of non-collagenous proteins, a more rapid and more intensive wetting of the hide is assured. Advantageously, the soak water is made alkaline (see supra), but the pH value should always remain less than 12. Additionally, soaking auxiliaries such as nonionic and anionic surfactants in combination with substituted phenols or dithiocarbamates in the usual concentration ranges (0.1-10 g/l) are advantageous. As enzymatic additives in the enzyme formulations according to the invention, the aforementioned proteases, for example, particularly the proteases mentioned under c) come into consideration, especially microbial proteases in the active range of 7-11.0, particularly Bacillus proteases, Streptomvces proteases, as well as fungal proteases, e.g. from Aspergillus species like A.saitoi and A.usamii, also such as from A.oryzae having a activity region of pH 7.0-9.5, and also from A.niger and A.flavus having a range of pH effectiveness at 9.5-11.0.
In general, the concentration of proteolytic activities of the proteinases employed is in the range from 0.1-0.3 Anson units, or 10,000 to 30,000 Lxc3x6hlein-Volhard units per liter of soaking bath. The amounts of enzyme preparation added thus correspond to these concentrations, depending on the measure of their enzyme content. Finally, the soaking baths may additionally contain amylases. The amylases occur, e.g., as accompanying enzymes of fungal proteases. They improve the cleavage of glycosidic bonds in the proteoglycans and glycoproteins of the hide.
In the processes of the beamhouse, liming as a rule follows the soak, followed by deliming and thexe2x80x94predominantly enzymaticxe2x80x94bate.
The enzyme preparation can also be employed to advantage in these subsequent steps.
Use: Bating
Deliming traditionally serves to reduce the alkalinity of the unhaired hides from pH values of 13-14 to pH values in the region from 7-8. For deliming, strongly dissociated acids should preferably not be used, but rather weak organic acids, e.g. of the dicarboxylic acid type, or weakly acid salts. In bating, residues of epidermis, hair, and pigment are to be removed and an additionally opening of the hide effected. Further, non-collagenous protein residues are removed (cf. Ullmann, op.cit., 4th edition, volume 16, pages 119-120). Bating conventionally proceeds at a pH of 7.5 to 8.5. The use of cyclic carbonates in deliming is known from DE-A 3,108,428.
The amount of protease employed in the bate liquor is from 5,000 to 20,000 Lxc3x6hlein-Volhard units per liter.
Lipases can concurrently be used in the bate, for example pancreas lipases having an activity region at pH 7.0-9.0. Also amylases according to A., for example pancreas amylases, having an activity region at pH 5.5-8.5, which favor the cleavage of glycosidic bonds in the bate, have a favorable influence on bating (above all as accompanying enzymes of trypsin and chymotrypsin).
The following Examples serve to illustrate the invention.